Transfection of short hairpin RNA (shRNA) expression plasmids is conventionally performed for gene-specific knockdown in cultured mammalian and insect cells. Here, I describe a simple method to synthesize an inverted repeat DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded inverted repeat DNA and Bst DNA polymerase. The shRNA expression plasmids constructed by this method were confirmed to promote efficient RNA interference knockdown in silkworm cell lines. This method may be useful for constructing a relatively large number of shRNA expression plasmids.